CN105154342A - Method for cultivating liquid-state morchella strain - Google Patents
Method for cultivating liquid-state morchella strain Download PDFInfo
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- CN105154342A CN105154342A CN201510704225.XA CN201510704225A CN105154342A CN 105154342 A CN105154342 A CN 105154342A CN 201510704225 A CN201510704225 A CN 201510704225A CN 105154342 A CN105154342 A CN 105154342A
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Abstract
The invention provides a method for cultivating a liquid-state morchella strain. The method includes the steps that a liquid-state morchella strain culture medium is provided; a stock morchella strain is inoculated into the liquid-state morchella strain culture medium for conducting airtight culture; after hyphae of the stock morchella strain germinate, the liquid-state morchella strain culture medium is shaken once every day till a morchella cultivated species is obtained. According to the method for cultivating the liquid-state morchella strain, operation is easy, the cultivated hyphae of the morchella cultivated species grow fast, the produced hyphae are even and neat, contamination by other strains is small, and the artificial cultivation survival rate and yield of morchella are increased; moreover, the culture process is simple and easy to implement, and cost is low.
Description
Technical field
The present invention relates to biological technical field, specifically, relate to a kind of cultural method of morel liquid strain.
Background technology
Morel is the rare wild edible fungus of a kind of preciousness, is of high nutritive value.Though have the artificial culture reporting and realize morel at present, but the morel spawn culture based component adopted often produces considerable influence to the growth quality of morel bacterial classification, need through multiple steps such as inoculation, quiescent culture and shake-flask culture in morel spawn culture process, but also need manually to pass into sterile air in described culture vessel, cause that culturing step is loaded down with trivial details, labour intensity is high.Therefore, design the arrange in pairs or groups cultivation of cultural method to morel of rational morel liquid strain substratum and the simple morel liquid strain of a kind of culture process of a kind of composition to be of great immediate significance.
In order to solve above Problems existing, people are seeking a kind of desirable technical solution always.
Summary of the invention
The object of the invention is for the deficiencies in the prior art, a kind of cultural method of morel liquid strain is provided.
To achieve these goals, the technical solution adopted in the present invention is a kind of cultural method of morel liquid strain, specifically comprises the following steps:
A kind of morel liquid strain substratum is provided;
Morel original seed is inoculated in described morel liquid strain substratum and carries out sealing cultivation, after the mycelium germination of described morel original seed, shake described morel liquid strain substratum every day 1 time until obtain morel cultivar.
Preferably, the step of described morel liquid strain substratum is provided to comprise:
Pine branch 200-300g, potato 200-400g and water 1200ml-1800ml are mixed, then successively through boiling, cool, filtration treatment obtains filtrate, glucide 15-30g is joined in described filtrate and also dissolve obtained morel liquid strain nutrition filtrate, get described morel liquid strain nutrition filtrate 150ml and be placed in culturing bottle, then in described culturing bottle, the husk 10-30g and calcium carbonate 1-5g that clean and dry is added, obtained morel liquid strain substratum.
Based on above-mentioned, prepare in described morel liquid strain nutrition filtrate step, described glucide refers to sucrose, glucose or maltose etc., preferably, be soaked in water fresh wheat 300-400g, and at room temperature impel described malting until Fructus Hordei Germinatus length is about the 3-6 of described wheat length doubly, then, described malt drying is worn into malt meal, then in described malt meal, the described filtrate of described malt meal 5-7 times quality is added, obtained morel liquid strain nutrition filtrate after saccharification, filtration.
Preferably, after described morel original seed mycelium germination, shook described morel liquid strain substratum 1 time at interval of 24 hours, the husk in described morel liquid strain substratum is soaked completely.In brief, in the training period, every day, timing shook described morel liquid strain substratum 1 time, such as every morning 8 point, every morning 10 or shake every afternoon 3 described morel liquid strain substratum 1 time, make husk be immersed in described morel liquid strain substratum completely medium.
Preferably, described morel original seed obtains by carrying out one-level cultivation or secondary to cultivate to morchella mother culture.
Based on above-mentioned, the step that described morchella mother culture carries out one-level cultivation comprises:
Cotton seed hulls 20-30g, weed tree sawdust 15-20g, husk 20-30g, wheat bran 15-25g, Semen Maydis powder 8-12g are soaked in water respectively, filtration is pulled out and is added water boil respectively afterwards, pull out and drain, then carry out mixing obtained compound, in described compound, add sucrose 5-10g, calcium carbonate 1-5g and phosphate fertilizer 1-5g, mix obtained morel one-level pedigree seed culture medium;
High-temperature sterilization, cooling process are carried out to described morel one-level pedigree seed culture medium, then in described morel one-level pedigree seed culture medium, accesses morchella mother culture and cultivate, obtain morel one-level original seed.
Based on above-mentioned, wherein prepare in morel one-level pedigree seed culture medium process, described cotton seed hulls, weed tree sawdust are soaked 30-35 hour, and every 5 hours change water once, described husk soaks 3-4 hour, and described wheat bran, Semen Maydis powder soak 1-3 hour.
Based on above-mentioned, the step that described morchella mother culture carries out secondary cultivation comprises:
Pine needle 100-200g, needle mushroom 100-200g and water 1200ml-1800ml are carried out mixing, boiling, cooling, filtration treatment, obtained morel secondary original seed nutritive medium, then wheat 20-30g, corn 25-40g, husk 15-25g, rice bran 15-25g, sucrose 1-5g, gypsum 1-5g, phosphate fertilizer 1-5g are mixed obtained morel secondary pedigree seed culture medium compound, and join with the described morel secondary original seed nutritive medium of the quality such as described morel secondary pedigree seed culture medium compound, mix obtained morel secondary pedigree seed culture medium;
Described morel secondary pedigree seed culture medium is carried out high-temperature sterilization, cooling process, then morel one-level original seed is linked in described morel secondary pedigree seed culture medium and cultivates, obtain morel secondary original seed.
Based on above-mentioned, the cultural method of described morchella mother culture comprises the steps:
Potato 50-60g, agar 8-14g, vitamins B agent 0.5-0.8g and glucose 5-7g are added in the water of 1500ml and heats, until described agar all boiling, obtain morchella mother culture substratum; High-temperature sterilization, cooling process are carried out to described morchella mother culture substratum, then morel bacterial strain mycelia is linked into described morchella mother culture substratum and cultivates, obtain described morchella mother culture.
Hinge structure of the present invention has outstanding substantive distinguishing features and significant progress, specifically,
Morel liquid strain cultural method provided by the present invention, in the process that morel liquid strain is cultivated, do not need to shake continuously substratum, only need every day to the shake of morel liquid strain substratum once, reduce the utilization ratio of the equipment that culturing process utilizes, thus reduce production cost, and this cultural method is simple, easy handling; Timing shake is adopted every day to ensure that the homogeneity of described morel liquid strain mycelia nutritive medium.Further, the growth of Morciiella Esculeuta Mycelia take husk as carrier, the gap between husk is utilized to absorb nutrition, and be attached on the husk on liquid surface and grow, without the need to additionally passing into sterile air in its substratum, decrease the sterile equipment of numerous and diverse operation and many costlinesses, saved cost and shortened cultivation time of bacterial classification.
Further say, the raw material of the liquid strain of morel described in the present invention substratum re-uses after liquor, makes the nutriment distribution of bacterium culture medium relatively more even, is conducive to Morchella esculenta (L.) Pers mycelium and absorbs, the bacterial classification straight uniform of output.Adopt different culture medium raw materials in each stage of breeding strain, the morel of different cultivation stage fully can be absorbed the nutritive ingredient in substratum, reach best way to cultivate.
Accompanying drawing explanation
Fig. 1 is the growth curve chart of No. 926, Morchella crassipes in embodiment 1.
Embodiment
Below by embodiment, technical scheme of the present invention is described in further detail.
Embodiment 1
The present embodiment provides a kind of cultural method of morel liquid strain, selects Morchella crassipes No. 926 original seeds to cultivate as the bacterial strain of morel cultivar, specifically comprises the following steps:
Pine branch 200g, potato 200g and water 1800ml are mixed, boil and keep 30 minutes, is then cooled to room temperature, obtained filtrate after filtering; Sucrose 30g is joined in described filtrate and also dissolve obtained morel liquid strain nutrition filtrate, get described morel liquid strain nutrition filtrate 150ml and be placed in culturing bottle, then in described culturing bottle, the husk 20g and calcium carbonate 5g that clean and dry is added, obtained morel liquid strain substratum.
Described morel liquid strain substratum is positioned in Autoclave, at 1.5kg/cm3, sterilizing 0.5 hour at 110 DEG C, after taking-up is cooled to room temperature, the triangular flask that described morel liquid strain substratum is housed is placed in inoculation tank sealing 2 hours, then Morchella crassipes No. 926 original seeds are linked in described morel liquid strain substratum, the culturing room being placed in 30 DEG C carries out sealing cultivation, when after described morel original seed mycelium germination, the husk that timing o'clock sharp every morning 10 is shaken in described substratum 1 time to described substratum is soaked completely, until obtain Morchella crassipes No. 926 cultivars.
In order to better understand the growing state of different times Morchella esculenta (L.) Pers mycelium, determining liquid incubation time, is within the scope of 0-144h at growth time, utilizes the every 12h of special sampling thief to carry out 1 sub-sampling, measures Morciiella Esculeuta Mycelia biomass.Concrete operation step is: the cultivation of morel bacterial classification per stage selects sampling thief to extract liquid appropriate in described triangular flask after terminating, take rotating speed as 4000r/min centrifugation 15min, then clear liquid is discarded, the Morchella esculenta (L.) Pers mycelium distilled water obtained is cleaned for several times, be placed in vacuum drying oven, dry at 40 DEG C to constant weight, calculate the Morciiella Esculeuta Mycelia biomass cultivated and obtain.Often group carries out 3 parallel tests, and the data obtained gets the mean value of 3 test-results, take incubation time as X-coordinate, and Morciiella Esculeuta Mycelia biomass is ordinate zou, draws the growth curve of morel.
Calculation formula is as follows:
Y=M/V
In formula: Y represents Morciiella Esculeuta Mycelia biomass, unit g/L;
M represents the dried quality of mycelium, unit g;
V represents the volume of extracting liquid, unit L.
Gained liquid state cultivates morel growth curve as shown in Figure 1, as can be seen from the figure, liquid cultivate morel growth curve can be divided into adjustment period, logarithmic phase, stationary phase and decline phase these four periods, the Morciiella Esculeuta Mycelia biomass of the morel cultivar of gained is 13.95g/L, and production cost is 0.326 yuan/g; Average growth rate is 1.0cm/d, and covering with the substratum time is 5 days, and without varied bacteria growing; And the morel cultivar hypha biomass of quiescent culture gained is 9.241g/L.
Thinking, in culturing process, by fully rocking described liquid strain substratum, dissolved oxygen amount in described liquid strain substratum significantly being increased; In addition, because mycelium depends on husk growth, rock make nutritive ingredient evenly, thus make Morchella esculenta (L.) Pers mycelium growth metabolism vigorous, it is high to produce output.
Embodiment 2
The present embodiment provides a kind of cultural method of morel liquid strain, Morchella crassipes No. 926 original seeds are selected to cultivate as the bacterial strain of morel cultivar, concrete culturing step is as follows: pine branch 300g, potato 400g and water 1800ml are mixed, boil and keep 30 minutes, then room temperature is cooled to, obtained filtrate after filtering; Get fresh wheat 400g, soak 12 hours in clear water, it is allowed to germinate under room temperature, when its bud is about 3 times into wheat, take out and dry at 60-80 DEG C, wear into malt meal, then described malt meal is added in described filtrate, the described filtrate added is 6 times of described malt meal, saccharification 6 hours obtained morel liquid strain nutrition filtrate after 4 layers of filtered through gauze under the condition of 50 DEG C; Get described morel liquid strain nutrition filtrate 150ml and be placed in culturing bottle, in described culturing bottle, then add the husk 20g and calcium carbonate 5g that clean and dry, obtained morel liquid strain substratum.
Described morel liquid strain substratum is positioned in Autoclave, at 1.5kg/cm3, sterilizing 0.5 hour at 110 DEG C, after taking-up is cooled to room temperature, the triangular flask that described morel liquid strain substratum is housed is placed in inoculation tank sealing 2 hours, then Morchella crassipes No. 926 original seeds are linked in described morel liquid strain substratum, the culturing room being placed in 30 DEG C carries out sealing cultivation, when after described morel original seed mycelium germination, the husk that timing o'clock sharp every morning 10 is shaken in described substratum 1 time to described substratum is soaked completely, until obtain Morchella crassipes No. 926 cultivars.
It is mean value is 8.545g/L that experiment records the biomass of No. 926, Morchella crassipes adopting this liquid strain culture medium culturing to go out, and production cost is 0.355 yuan/g.
Embodiment 3
The present embodiment provides a kind of cultural method of morel liquid strain, Morchella crassipes No. 926 original seeds are selected to cultivate as the bacterial strain of morel cultivar, as different from Example 1, the culture medium raw material adopted in the culturing process of described Morchella crassipes No. 926 cultivars is as follows: wheat bran 40g, Semen Maydis powder 20g, analysis for soybean powder 10g, KH2PO41g, MgSO41g, it be mean value is 7.148g/L that experiment records the biomass of No. 926, Morchella crassipes adopting this liquid strain culture medium culturing to go out, and production cost is 0.465 yuan/g.
Embodiment 4
The present embodiment provides a kind of cultural method of morel liquid strain, Morchella crassipes No. 926 original seeds are selected to cultivate as the bacterial strain of morel cultivar, with embodiment 1, embodiment 2 and embodiment 3 all unlike, the culture medium raw material adopted in the culturing process of described Morchella crassipes No. 926 cultivars is as follows: potato 200g, sucrose 30g, yeast extract paste 5g, KH2PO40.5g, MgSO40.5g, it be mean value is 6.754g/L that experiment records the biomass of No. 926, Morchella crassipes adopting this liquid strain culture medium culturing to go out, and production cost is 0.41 yuan/g.
Embodiment 5
The present embodiment provides a kind of cultural method of morel liquid strain, and as different from Example 1, the present embodiment selects Morchellaconica No. 405 bacterial strains as morel to cultivate, and concrete culturing step comprises:
(1) cultivation of No. 405, Morchellaconica mother kind
Water potato 60g, agar 10g, vitamins B agent 0.5g and glucose 5g being put into 1500ml be heated to agar all boiling namely obtain morchella mother culture substratum.
Get 150ml morchella mother culture substratum to be positioned in the test tube of 250ml and to seal with cotton, described test tube to be positioned in the pressure kettle at 130 DEG C sterilizing 1 hour, then room temperature is naturally cooled to, the described test tube that described morchella mother culture substratum is housed is put into the aseptic inoculation box of sealing, and in described test tube, access Morchellaconica No. 405 bacterial strain mycelia, being placed in temperature is that the incubator of 30 DEG C is cultivated, and plants until grow No. 405, Morchellaconica mother.
(2) cultivation of Morchellaconica No. 405 one-level original seeds
Cotton seed hulls 30g, weed tree sawdust 20g are soaked in water 30 hours and changed water once every 5 hours; 20g husk is soaked 3 hours; Wheat bran 25g, Semen Maydis powder 8g are soaked in water 3 hours respectively; Then soaked cotton seed hulls, wheat bran, weed tree sawdust, husk, Semen Maydis powder are placed in respectively pot and add water boil 2 hours, then filter, drain; Then in the described cotton seed hulls after draining, wheat bran, weed tree sawdust, husk, Semen Maydis powder, add sucrose 10g, calcium carbonate 1g and phosphate fertilizer 5g, mix obtained morel one-level pedigree seed culture medium.
Described morel one-level pedigree seed culture medium to be loaded in the wide-necked bottle of 100ml and to seal with kraft paper, it is 40g that every bottle of Intake Quantity amounts to dry weight, point wide-necked bottle installed is sealed a bottle hole with the polypropylene duplicature of 10 ㎝ × 10 ㎝, cut off facing to wide-necked bottle position the hole that a diameter is 1.5 ㎝ at polypropylene duplicature, and seal with two-layer kraft paper, with the fixing sealing of cotton rope; By the sterilizing 3 hours at 120-130 DEG C of the wide-necked bottle after sealing, then naturally cooling in sterilisable chamber; Planting No. 405, Morchellaconica mother according to every bottle of inoculum size is that 4g is inoculated in described morel one-level pedigree seed culture medium, and being positioned over temperature is cultivate in the thermostat container of 30 DEG C, cultivates and obtains Morchellaconica No. 405 one-level original seeds in 10 days.
(3) cultivation of Morchellaconica No. 405 secondary original seeds
Pine needle 200g, needle mushroom 200g and 1800ml are mixed, boil and keep 20 minutes, cooled and filtered obtains nutrition filtrate, then take wheat 20g, corn 25g, husk 15g, rice bran 15g, sucrose 1g, gypsum 1g, phosphate fertilizer 1g respectively, above-mentioned raw materials component is mixed and obtains compound.
The described morel secondary pedigree seed culture medium filtrate of equal quality is added in described compound, mix system, and be divided into some equal portions and be respectively charged in multiple triangular flask and seal with cotton, be positioned in Autoclave, in 2kg/cm3, sterilizing 1 hour at 110 DEG C, take out and be cooled to room temperature and obtain morel secondary pedigree seed culture medium.
Multiple described triangular flask is placed in inoculation tank sealing 1 hour, Morchellaconica No. 405 one-level original seeds are linked in described morel secondary pedigree seed culture medium, and send into culturing room, the temperature controlling culturing room is 30 DEG C, cultivates and obtains Morchellaconica No. 405 secondary original seeds in 15 days.
(4) cultivation of Morchellaconica No. 405 morel cultivars
Pine branch 300g, potato 300g and water 1800ml are mixed, boil and keep 30 minutes, is then cooled to room temperature, obtained nutrition filtrate after filtering.
Sucrose 25g is joined in described filtrate and also dissolve obtained morel liquid strain nutrition filtrate, get described morel liquid strain nutrition filtrate 150ml and be placed in culturing bottle, then in described culturing bottle, the husk 20g and calcium carbonate 5g that clean and dry is added, obtained morel liquid strain substratum.
The triangular flask that described morel liquid strain substratum is housed is positioned in Autoclave, 1.5kg/cm3, sterilizing 1 hour at 120 DEG C, taking-up is cooled to room temperature, then inoculation tank sealing 3 hours is placed in, be linked in described morel liquid strain substratum by Morchellaconica No. 405 secondary original seeds, the culturing room being placed in 30 DEG C carries out sealing cultivation, when after described morel secondary original seed mycelium germination, fully shook 1 time at interval of 1 day, cultivate and obtain Morchellaconica No. 405 cultivars in 10 days.
The average growth rate that experiment records No. 405, the Morchellaconica adopting this liquid strain culture medium culturing to go out is 0.7cm/d, and covering with the substratum time is 7 days, and without varied bacteria growing.
Embodiment 6
The present embodiment provides a kind of cultural method of morel liquid strain, as different from Example 5, the present embodiment selects delicious morel No. 918 bacterial strains as morel cultivar to cultivate, concrete outcome is as follows: the average growth rate that experiment records the delicious morel No. 918 adopting this liquid strain culture medium culturing to go out is 0.8cm/d, covering with the substratum time is 6 days, and without varied bacteria growing.
Finally should be noted that: above embodiment is only in order to illustrate that technical scheme of the present invention is not intended to limit; Although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the field are to be understood that: still can modify to the specific embodiment of the present invention or carry out equivalent replacement to portion of techniques feature; And not departing from the spirit of technical solution of the present invention, it all should be encompassed in the middle of the technical scheme scope of request of the present invention protection.
Claims (8)
1. a cultural method for morel liquid strain, comprises the following steps:
A kind of morel liquid strain substratum is provided;
Morel original seed is inoculated in described morel liquid strain substratum and carries out sealing cultivation, after the mycelium germination of described morel original seed, shake described morel liquid strain substratum every day 1 time, until obtain morel cultivar.
2. the cultural method of morel liquid strain according to claim 1, is characterized in that, provides the step of described morel liquid strain substratum to comprise:
Pine branch 200-300g, potato 200-400g and water 1200ml-1800ml are mixed, then successively through boiling, cool, filtration treatment obtains filtrate;
Glucide 15-30g is joined in described filtrate and also dissolve obtained morel liquid strain nutrition filtrate, get described morel liquid strain nutrition filtrate 150ml and be placed in culturing bottle;
The husk 10-30g and calcium carbonate 1-5g that clean and dry is added, obtained morel liquid strain substratum in described culturing bottle.
3. the cultural method of morel liquid strain according to claim 2, is characterized in that,
The step preparing described morel liquid strain nutrition filtrate comprises: be soaked in water fresh wheat 300-400g, and at room temperature impel described malting until Fructus Hordei Germinatus length is about the 3-6 of described wheat length doubly, then, described malt drying is worn into malt meal, and in described malt meal, add the described filtrate of described malt meal 5-7 times quality, obtained morel liquid strain nutrition filtrate after saccharification, filtration.
4. the cultural method of the morel liquid strain according to any one of claim 1-3, it is characterized in that, after described morel original seed mycelium germination, shook described morel liquid strain substratum 1 time at interval of 24 hours, the husk in described morel liquid strain substratum is soaked completely.
5. the cultural method of morel liquid strain according to claim 4, is characterized in that, described morel original seed obtains by carrying out one-level cultivation or secondary to cultivate to morchella mother culture.
6. the cultural method of morel liquid strain according to claim 5, is characterized in that, the step that described morchella mother culture carries out one-level cultivation comprises:
Cotton seed hulls 20-30g, weed tree sawdust 15-20g, husk 20-30g, wheat bran 15-25g, Semen Maydis powder 8-12g are soaked in water respectively, after filtration, add water boil respectively, filtration drains, then mix, obtained compound; In described compound, add sucrose 5-10g, calcium carbonate 1-5g and phosphate fertilizer 1-5g, mix obtained morel one-level pedigree seed culture medium;
High-temperature sterilization, cooling process are carried out to described morel one-level pedigree seed culture medium, then in described morel one-level pedigree seed culture medium, accesses morchella mother culture and carry out one-level cultivation, obtain morel one-level original seed.
7. the cultural method of morel liquid strain according to claim 6, is characterized in that, the step that described morchella mother culture carries out secondary cultivation comprises:
Pine needle 100-200g, needle mushroom 100-200g and water 1200ml-1800ml are carried out mixing, boiling, cooling, filtration treatment, obtained morel secondary original seed nutritive medium, then wheat 20-30g, corn 25-40g, husk 15-25g, rice bran 15-25g, sucrose 1-5g, gypsum 1-5g, phosphate fertilizer 1-5g are mixed obtained morel secondary pedigree seed culture medium compound, and join with the described morel secondary original seed nutritive medium of described morel secondary pedigree seed culture medium compound equal in quality, mix obtained morel secondary pedigree seed culture medium;
High-temperature sterilization, cooling process are carried out to described morel secondary pedigree seed culture medium; Then described morel one-level original seed is linked in described morel secondary pedigree seed culture medium and carries out secondary cultivation, obtain morel secondary original seed.
8. the cultural method of morel liquid strain according to claim 5, is characterized in that, the cultural method of described morchella mother culture comprises the steps:
Potato 50-60g, agar 8-14g, vitamins B agent 0.5-0.8g and glucose 5-7g are added in the water of 1500ml and heats, until described agar all boiling, obtain morchella mother culture substratum;
High-temperature sterilization, cooling process are carried out to described morchella mother culture substratum; Then morel bacterial strain mycelia is linked in described morchella mother culture substratum and cultivates, obtain described morchella mother culture.
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| CN107805103A (en) * | 2017-11-29 | 2018-03-16 | 穆世鹏 | A kind of edible fungus culture medium |
| CN111903432A (en) * | 2020-08-06 | 2020-11-10 | 贵州珍稀食用菌科技有限公司 | Dictyophora rubrovalvata strain rapid propagation method and application thereof |
| US11116807B2 (en) * | 2017-12-13 | 2021-09-14 | Grape King Bio Ltd. | Active substance of Morchella, its use and a composition thereof for improving the reproductive function |
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| CN111903432B (en) * | 2020-08-06 | 2022-04-01 | 贵州珍稀食用菌科技有限公司 | Dictyophora rubrovalvata strain rapid propagation method and application thereof |
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